PT2236598E - Microorganisms for improving the health of individuals with disorders related to gluten ingestion - Google Patents

Description

ΕΡ2236598Β1

DESCRIPTION

GLUTEN INDIVIDUALS

MICRORGANISMS TO IMPROVE THE STATE OF HEALTH WITH DISORDERS RELATED TO THE INGESTION OF

TECHNICAL FIELD The invention relates to the Food Industry and Pharmaceutical sectors. Specifically, the invention relates to the field of probiotics and by-products in the form of functional foods and novel foods, probiotics, symbiotics, nutraceuticals or food supplements and pharmaceutical compositions for clinical applications.

STATE OF THE ART Celiac disease is an enteropathy, an intestinal disorder, of an immune nature caused by permanent intolerance to cereal gluten proteins, which affects genetically predisposed individuals. The disease has a broad clinical spectrum and includes typical, atypical, silent, and potential forms. The typical forms most frequently present in the first years of life (6-24 months) and manifests mainly with intestinal symptoms and related alterations (malabsorption, chronic diarrhea, weight loss, abdominal distension, growth retardation, etc.). It is usually the most common chronic disease, with a prevalence of 0.7 to 2.0% among the general population and 15 to 20% among first-degree relatives. Also, gluten intake and celiac disease are associated with other disorders such as Down's Syndrome, Type 1 diabetes mellitus, herpetiform dermatitis, myopathy, multiple sclerosis, arthritis, autism, schizophrenia, depression, lymphomas, and ataxia. The relationship between gluten intake and psychiatric, neurological and behavioral disorders is considered a result of the production of bioactive peptides, such as exorphins, having opioid activity.

Gluten proteins (gliadins and prolamins and analog glutenins) represent the major environmental factor that triggers celiac disease and other related disorders. These proteins contain peptide sequences rich in proline and glutamine, making them more resistant to digestive enzymes than other proteins in the diet and thus able to remain in the intestinal lumen. In genetically predisposed individuals, these peptides are responsible for an analogous reaction involving the innate and adaptive immune system and which, overall, leads to chronic inflammation of the intestinal mucosa, increased intraepithelial lymphocytes, crypt hyperplasia, and progressive deterioration of intestinal villi including its total disappearance. The toxic peptides produced after the ingestion of gluten can pass through the intestinal epithelium and are recognized by the HLA-DQ2 or HLA-DQ8 molecules of antigen presenting cells, preferably after their deamidation through the action of tissue transglutaminase. In this way, these are presented to T-cell receptors, producing their activation. This involves the expression of the CD4 antigen, also known as helper (Th), and its differentiation into subpopulations of lymphocytes, thereby involving acquired immunity. The Th2 subpopulation interacts with B cells that differentiate into plasma cells and produce anti-gliadin, antiendomysium, and anti-tissue transglutaminase antibodies. The Th1 subpopulation is responsible for an increase in the secretion of proinflammatory cytokines (mainly IFN-γ) and in the proportion of IFN-γ / IL-10 (Salvati et al 2005. Recombinant human interleukin 10 suppresses gliadin dependent T cell activation in ex live cultured coeliac intestinal mucosa Gut 54: 46-53). Gluten peptides may also elicit a response in the intestinal epithelium mediated by the IL-15 cytokine, involving the innate immune system (Green and Jabri 2006, Celiac disease, Ann Rev Med 57: 207-21). Ingested gliadins stimulate IL-15 production in epithelial cells, which favors the clonal expansion of intraepithelial cytotoxic CD8 T lymphocytes and the expression of IFN-γ (Jabri et al., 2000). Selective expansion of intraepithelial lymphocytes expressing the natural HLAE-specific killer receptor CD94 in celiac disease Gastroenterology 118: 867-79 Mention et al., 2003 Interleukin 15: a key to disrupted intraepithelial lymphocyte homeostasis and limphomagenesis in celiac disease Gastroenterology 125: 730-45 Forsberg et al. 2007. Concomitant increase of IL-10 and pro-inflammatory cytokine in intraepithelial limphocyte subsets in celiac disease Int Immunol 2007: 19, 993-1001). The intestinal microbiota composition of patients with celiac disease is also imbalanced relative to that of healthy control subjects, characterized by the predominance of pro-inflammatory bacteria and a reduced proportion and composition of lactic acid and bifidobacteria bacteria. (Sanz et al., 2007. Differences in Faecal Bacterial Communities in Coeliac and Healthy Children as Detected by PCR and denaturing gradient gel electrophoresis FEMS Immunol Med Microbiol 51 (3): 562-8. in the composition of the duodenal microbiota of children with 3 ΕΡ 2236598Β1 celiac disease.J Med Microbiol 2007 Dec; 56 (Pt 12): 1669-74; Collado et al., 2009. Specific duodenal and faecal bacterial groups are associated with pediatric celiac disease J Clin Pathol, 62: 264-269). In the intestinal lumen and epithelium, the combination of gluten with an increase in harmful bacteria may act as a triggering factor or favor the pathological process and pro-inflammatory reactions in cases of active celiac disease, as well as in other related disorders. Likewise, the presence or absence of certain bacterial species may favor or protect against the toxicity of gluten. Celiac disease has a high incidence and severity; however, there is currently no therapy for these patients. The only alternative is to maintain a strictly gluten-free diet of long duration. Compliance with this dietary recommendation is difficult and patients continue to suffer gastrointestinal symptoms, nutritional deficiencies, and high health risks (immune disorders, osteoporosis, infertility, cancer, etc.) and the balance of their intestinal ecosystem is never fully restored . In addition, individuals with refractory celiac disease (5-10%) do not respond to this dietary recommendation.

Therapeutic or coadjuvant options that are normally under investigation for treating celiac disease include oral administration of proteolytic enzymes obtained from plants or microorganisms to accelerate gastrointestinal digestion of gluten peptides (Shan et al., 2005). Enzyme treatment of foodstuffs for celiac (2006), pp. 20050249719 / A1, Marti et al., 2006 Prolyl endopeptidase mediated destruction of T cell epitopes in whole gluten 4 ΕΡ2236598Β1 20060286601 / Α1, Stepniak and Koning, 2006. Enzymatic gluten detoxification: the proof of the pudding is in the eating! Trends Biotechnol 24: 433-4 Gass et al.

enzyme therapy for gastric digestion of dietary gluten in patients with celiac sprue. Gastroenterology. 133: 472-80). The effectiveness of this strategy has been demonstrated in model systems using protein and peptide preparations or their recombinant equivalents, but studies demonstrating their in vivo efficacy are still required in subjects who ingest gluten as in food. Despite the potential benefits of this therapy as an adjuvant in a gluten-free diet, its effects will be highly dependent on the ingestion time of the enzyme preparation and will only allow the occasional intake of gluten reducing the threshold of toxicity. Other proposed alternatives include the development of tissue transglutaminase inhibitor compounds (Khosla et al., 2006, Transglutaminase inhibitors and methods of use thereof W02007025247), antibodies capable of capturing gliadin peptides (Fox, 2007. Antibody therapy for treatment of diseases associated Gluten intolerance, 20070184049 / A1), compounds that block the binding sites of gluten peptides to HLA-DQ2 or HLA-DQ8 molecules (Sollid et al., 2007; Peakman and Chicz. 2007. Peptide epitopes recognized by disease promoting CD4 + T limphocytes. 20070142622 / A1), pro-inflammatory cytokine antagonists such as IFN-γ (Maroun et al., 2007), oral administration of recombinant regulatory cytokines (Salvati et al. , 2005, 54: 46-53), 5 ΕΡ2236598Β1 inhibitors of adhesion molecules involved in the inflammatory reactions, and zonulin antagonists responsible for the disease by increases in paracellular permeability (Paterson and Ginski, 2007. Formulations for a tight junction effector US20070196501 / A1). These strategies cause the modification of molecules involved in various biological processes and their use can lead to unwanted side effects. Food industry strategies are being developed to avoid the presence of toxic epitopes in food that is ingested through the genetic manipulation of certain wheat varieties and the use of proteolytic enzymes and lactobacilli during the fermentation processes of cereals that may degrade the toxic epitopes. Thus, the goal is to introduce improvements in the diet of celiac patients and to provide them with a wide variety of products but without preventing or treating the disease itself (Rizzello et al., 2007). Highly efficient gluten degradation by lactobacilli and fungai proteases during food Processing: new perspectives for celiac disease, Appl Environ Microbiol.73: 4499-507). The use of strains belonging to the genus Bifidobacterium as probiotics or pharmaceutical preparations for the treatment and prevention of celiac disease and related disorders is the basis of the present invention. The benefits of specifically selected bifidobacteria for this purpose are numerous. Bifidobacteria have a special ability to colonize the intestinal tract of newborns, contributing significantly to the development of their (immune and other) defenses and oral tolerance to dietary antigens. This group of bacteria is one of the main constituents of the intestinal microbiota in the first years of life, particularly in breast fed children.

DESCRIPTION OF THE INVENTION

Brief Description of the Invention The application discloses a method for selecting microorganisms or microbial strains capable of modulating the immune response. These microorganisms, preferably belonging to the genus Bifidobacterium, are capable of being used for the treatment or prevention of immune mediated diseases such as celiac disease, owing to their immunomodulatory capabilities. The invention according to the claims provides a novel strain of the genus Bifidobacterium (CECT 7347; hereinafter " strain of the invention " (IATA-ES1) or the supernatant of a culture of the microorganisms of the invention or the extract obtained from the microorganisms of the invention in the form of preparations designed to reduce risks and improve the health and quality of life of individuals with celiac disease and other disorders related to gluten ingestion (allergy, autism, ataxia, diabetes, multiple sclerosis, etc.). strain of the invention has been isolated from feces of healthy breast fed infants and identified by sequencing of 16S rRNA and tuf genes. This strain has immunomodulatory properties capable of regulating proinflammatory responses characteristic of celiac disease and related diseases (sclerosis multiple, diabetes, 7α-2236598Β1 ataxia, etc.), as well as the Th2 type immune responses characteristics of IgE-mediated dietary protein allergies that result from the ingestion of wheat and other cereal proteins. The strain of the invention is characterized by inducing a low production of the cytokine IFN-γ Th1 and the pro-inflammatory cytokine IL-1, and by inducing a high production of IL-10 and TGF-β regulatory cytokines in peripheral blood mononuclear cells (PBMCs) . The cytokine profile induced by this strain is not a common feature of all the bifidobacteria and lactic acid bacteria of the human gut and makes it particularly suitable for modulating the abnormal immune response brought about by gluten proteins in predisposed individuals. Their combination with other microorganisms, such as for example B. longum ATCC15707, may potentiate the synthesis of the beneficial IL-10 regulatory cytokine for controlling the inflammation process characteristic of these pathologies. Non-viable bacteria (inactivated through and various procedures, such as heat, freeze-thaw, radiation, etc.) maintain their immunomodulatory properties. The strain of the invention is capable of incorporating and hydrolyzing the gluten peptides responsible for these disorders, reducing the concentration of toxic epitopes and their pathogenic potential. The strain of the invention has specific peptidases for hydrolyzing proline-containing substrates which are common to gluten proteins. Combinations of this with other strains of bifidobacteria that possess peptidases of diverse specificities allow their action to be complemented, favoring the degradation of toxic epitopes. The combination of bifidobacteria with other microorganisms such as Lactococcus lactis NCD0712, which has an εΡ2236598Β1 proteinase anchored in the cell wall, also potentiates the hydrolytic effects due solely to the action of bifidobacteria. The strain of the invention is able to modulate the immune response elicited by gluten peptides by: (i) inducing the synthesis of regulatory cytokines (IL-10), (ii) reducing the production of proinflammatory cytokines IFN-γ, IL -1, IL-8 and IL-15 derived from the innate and adaptive immune response (iii) inhibition of proinflammatory pathways mediated by nuclear factor kB (Example 3, Table 3). The strain of the invention as well as its derivative compounds according to the claims is also capable of inhibiting the pathogenic bacteria isolated from the intestinal microbiota of celiac disease patients with pro-inflammatory potential and virulence factors which favor the restoration of intestinal balance (Example 4, Tables 4 and 5). These strains are also able to inhibit the proinflammatory response of intestinal bacteria in patients with active celiac disease and those on a gluten-free diet, reducing the synthesis of proinflammatory cytokines (TNF-α and IFN-γ) and increasing of the regulatory cytokines (IL-10). The strain of the invention also has metabolic activities (for example: phosphatase, esterase, lipase, galactosidase, glucosidase and N-acetyl glucosaminidase) which promotes nutrient digestion and improves the malabsorption and malnutrition syndrome characteristic of celiac patients. has the ability to adhere to mucin (1-4%) and is stable under gastrointestinal stress conditions (acid pH and high bile concentration; Example 5, Table 6 and Table 7) and the technological processes of storage and preparation of (refrigeration, freeze-drying, fermentation, etc.). In vivo it is able to survive in transit in humans after oral administration. All these properties ensure their prolonged persistence and effectiveness in the gut and their use as probiotics and symbiotics (combinations of pro and prebiotics). These also ensure their use in the form of functional foods, novel foods, supplements, nutraceuticals and drugs to reduce risks and improve the health and quality of life of individuals with celiac disease as well as other disorders related to gluten intake.

Thus, a first aspect of the invention relates to a method for selecting microorganisms or strains of microorganisms capable of modulating the immune response to food allergies, which comprises: (a) isolating microorganisms from samples, preferably feces , from healthy individuals, preferably breast fed infants, and (b) selecting such prior stage microorganisms capable of modulating the immune response and hydrolyzing, inactivating or interfering with the mechanism of action of at least one agent causing food allergies, preferably celiac disease. Preferably, the microorganisms selected at stage (a) belong to a potentially probiotic species or genus. In a preferred embodiment, the immune response modulated by the microorganism obtained in stage (a) is the Th1 or Th2 proinflammatory responses. Also, the microorganisms obtained in stage (a) and (b), 10E-2236598Β1 alternatively or in addition to their ability to modulate the immune response, may be selected for their ability to increase viability and / or integrity of epithelial cells by enhancing function of intestinal barrier

The microorganisms selected by the above method or capable of hydrolyzing, inactivating or interfering with the mechanisms of action of at least one agent that causes food allergies and that modulates the immune response may be used for the treatment or prevention of food allergies (hereinafter " microorganisms of the invention "). Thus, one aspect of the invention relates to the claimed microorganisms for use as a medicament or food compositions, preferably for the treatment or prevention of food allergies. The invention relates to a bacterial strain with the deposition number CECT 7347. The invention further relates to a composition (hereinafter, the " composition of the invention ") which comprises the strain of the invention, wherein, in addition , is preferably present in this composition in a proportion between 0.1 and 99.9%, preferably between 1% and 99%, and more preferably between 10% and 90%. This composition may further comprise other microorganisms or media which potentiate, inter alia, the immunomodulatory capacity of the microorganism or strain of the invention or its ability to hydrolyze, inactivate or interfere with the mechanisms of action of the agents that cause food allergies. The invention further relates to a composition obtainable from the bioactive compounds derived from the microorganisms of the invention which are the supernatant of a culture of any of the microorganisms of the invention (hereinafter " supernatant of the invention "), or the extracts obtained from the pure or mixed culture of any of the microorganisms of the invention or strain of the invention (hereinafter " extract of the invention "), these bioactive compounds derived from the microorganism of the invention, hereinafter " bioactive compounds derivatives of the invention " can be used for the preparation of foods, supplements, nutraceuticals, probiotic or symbiotic products, novel foods or medicaments, and likewise their different uses are also part of the invention. Another aspect of the invention relates to a carrier material for the preparation of food products comprising the composition of the invention or at least one microorganism of the invention, preferably the strain of the invention. In a preferred embodiment, the microorganism of the invention is contained in the carrier material in an amount of at least about 105 cfu / g carrier material, preferably between 106 cfu / g and 1012 cfu / g, in a more preferred manner. preferred between 106 cfu / g and 1010 cfu / g. Accordingly, any food product or supplement comprising the composition of the invention is also part of the invention. The invention further relates to a pharmaceutical composition or food composition comprising any of the following compounds: the composition of the invention, the supernatant of the invention, the extract of the invention, a microorganism of the invention, the strain of the invention and optionally means and / or 12 Ρ 2236598Β1 pharmacologically acceptable excipients. The amount of microorganisms that the pharmaceutical composition or food composition will contain will vary according to the type of pathology it is designed for treatment. Preferably, said pathology is a food allergy, and, more preferably, celiac disease. In the case of the preparation of food compositions, and in accordance with the present invention, at least one microorganism of the invention or bioactive compound derived from the invention is incorporated into the carrier material, preferably in an amount of between 105 cfu / g and 1014 cfu / g of the carrier material, more preferably between about 108 cfu / g and 1013 cfu / g, and even more preferably between 107 cfu / g and 1012 cfu / g, in the case of a microorganism of the invention , and in the case of a bioactive compound derived from the invention in a proportion of between 0.1 and 99.9%, preferably between 1% and 99%, and more preferably between 10% and 90%. Such food compositions may be used for the preparation of nutraceuticals, functional foods, probiotics, symbiotics, nutritional supplements or any other types of product designed for the treatment or prevention, preferably of food allergies, and, more preferably, of celiac disease. The pharmaceutical composition or food composition of the invention may preferably be found in the form of tablets, capsules, microcapsules, powders, solutions, pastes, etc.

Another aspect of the invention relates to the composition of the invention, the supernatant of the invention, the extract of the invention, the pharmaceutical composition of the invention, or the feed composition of the invention wherein the microorganism of the invention or the strain of the invention is combined with another microorganism , a supernatant obtained from its culture or its subcellular fractions. Preferably, said microorganism belongs to the genus Lactococcus, preferably to the species Lactococcus lactis and, more preferably, is the strain Lactococcus lactis NCD0712. The invention also relates to the different forms of presentation of the composition of the invention which may be formulated as a food, nutraceutical, pharmaceutical preparation, supplement, probiotics or symbiotics, or novel foods.

Definitions:

Breast-fed infants: Throughout the disclosure, this term will preferably refer to healthy individuals, preferably less than two years of age, more preferably less than one year of age and, more preferably, less than 6 months of age, which were predominantly breast fed.

Healthy individuals: preferably this term refers to someone who does not suffer from any type of chronic or acute pathology according to the criteria of the medical specialists. Preferably, said pathology causes inflammation of the intestinal epithelium, more preferably celiac disease.

Food allergies: throughout the description this term will refer preferably to food allergies, preferably caused by milk, eggs, legumes, nuts, crustaceans, fish, molluscs, sesame, sunflower seeds, cottonseed , poppy seeds, beans, peas, lentils and, more preferably, gluten.

Thl-type pro-inflammatory response: the response to a stimulus which causes a high production of Th1-type cytokines, and preferably the cytokine IFN-γ which is preferably at least 100-fold, in a more preferably at least 15 times, more preferably at least 10 times, and still more preferably at least 4 times that of the control.

Pro-inflammatory response of the Th2 type: the response to a stimulus which causes a high production of Th2-type cytokines, and preferably the IL4 cytokine which is at least 100-fold, more preferably at least 15-fold, of a more preferred way, at least 10 times and even more preferably at least 4 times that of the control. Potentially probiotic genera or species: Preferably throughout the description, this term will refer to the species and strains of the following phylogenetic divisions and prokaryotic genera: Archaea, Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Verrucomicrobia, Fusobacteria, Spirochaetes, Fibrobacters, Deferribacteres, Deinococcus,

Thermus, Cyanobacteria, Methanobrevibacterium,

Bifidobacterium, Lactobacillus, Streptococcus, Leuconostoc. Peptostreptococcus, Pediococcus, Lactococcus, Enterococcus, Ruminococcus, Coprococcus, Subdolingranulum, Dorea, Bulleidia, Anaerobustis, Gemella, Roseburia, Catenibacterium, Dialister, 15 ΕΡ 2236598Β1

Anaerotruncus, Staphylococcus, Micrococcus, Propionibacterium, Enterobacteriaceae, Faecalibacterium, Bacteroid,

Parabacteroides, Prevotella, Eubacterium, Akkermansia, Bacillus, Butyrivibrio, Clostridium, etc. As well as the species and strains of fungi and yeasts Saccharomyces, Candida, Pichia, Debaryomyces, Torulopsis, Aspergillus, Rhizopus, Mucor, Penicillium, among others.

Means: This term refers preferably to culture media, substrates, prebiotics, fibers, bioactive compounds, excipients, ingredients, etc. which improve any features of the microorganisms of the invention for use in the preparation of medicaments, nutritional compositions, compositions, carrier materials, supernatants and foods of the invention (e.g., stability, immunomodulatory capabilities, adhesion capabilities, fermentation, hydrolysis or inactivation of allergy causing agents, etc.).

Support material: Preferably, the carrier material is a food composition selected from milk, yogurt, cheese, fermented milk, fermented milk-based food products, fermented cereals, flour, juices, sugar, cakes, ice creams , formulations for children, etc.

Description: This term will refer to pharmaceutical compositions or formulations, preferably designed to treat or prevent allergies, food allergies, inflammatory bowel diseases, gastrointestinal infections and the translocation of pathogenic microorganisms or their toxins, change of the intestinal balance (dysbiosis), bacterial overgrowth, change in intestinal permeability, food intolerance, celiac disease, malabsorption syndrome, etc.

Food Composition: Throughout the term this term will refer to foods (functional, conventional and novel), food supplements, nutritional formulas, and nutraceuticals, preferably designed to treat or prevent allergies, food allergies, inflammatory bowel diseases, gastrointestinal infections and translocation of pathogenic microorganisms or their toxins, altered intestinal balance (dysbiosis), bacterial overgrowth, altered intestinal permeability, food intolerance, celiac disease, malabsorption syndrome, etc.

A bioactive compound derived from a microorganism: any compound or molecule that forms part of the microorganism as a structural part, cellular component, subcellular fraction, metabolite or secreted molecule obtainable by physical chemical or biotechnological techniques including, inter alia, centrifugation, filtration, lyophilization, precipitation , sonication, mechanical and chemical cell disruption, extraction of the compound from cultures using enzymes and / or chemical agents, separation using chromatographic techniques, cloning and overexpression of genes encoding bioactive molecules that are capable of performing a beneficial function for the health. 17 ΕΡ 2236598Β1

DESCRIPTION OF THE FIGURES

Figure 1. SDS-PAGE analysis of the protein profiles of the different gliadin digestions. Panel A: 1) control of gliadins after digestion with pepsin (G-P); 2) control of gliadins after digestion with pepsin and trypsin (G-P-T); 3) G-P incubated with the strain of

invention; 4) G-P-T incubated with the strain of the invention (Bifidobacterium IATA-ES1). Panel B: 1) Control G-P; 2) control G-P-T; 3) G-P incubated with the strain of the invention and Lactococcus lactis NCD0712; 4) G-P-T incubated with the strain of the invention and Lactococcus lactis NCD0712.

Figure 2. Gram chromatogram obtained from the dialysable fraction of gliadin digestion in vitro. Peak 2 is that preferably hydrolyzed by the strain of the invention.

Figure 3. Alteration of cell viability (measured as endolysosomal activity) caused by the soluble and digested fraction of gliadins (Gld), in the presence or absence of bifidobacteria in vitro. Bovine albumin - BSA - (negative control); BifA2 (B. animalis); Bb (B. bifidum) and BL (Strain of the invention - Bifidobacterium longum IATA-ES1-). The results are expressed as mean values and the standard deviations are determined in quadruplicate.

Figure 4. Production of proinflammatory cytokines (TNF-a) and expression of nuclear factor kB (NF-kB) in cultures of Caco-2 cells exposed to the soluble and digested fraction of gliadins (Gld), in the presence or absence of

bifidobacteria in vitro. Bovine albumin - BSA 18 Ρ 2236598Β1 (negative control); BifA2 (B. animalis); Bb (B. bifidum) and BL (Strain of the invention - B. longum IATA-ES1-). The results are expressed as mean values and the standard deviations are determined in quadruplicate.

DETAILED DESCRIPTION OF THE INVENTION The present disclosure relates to a method for selecting microorganisms or strains of microorganisms capable of modulating the immune response in food allergies, which comprises: (a) isolating microorganisms from samples from healthy individuals, preferably infants fed (b) selecting the microorganisms from the previous step capable of modulating the immune response and hydrolyzing, inactivating or interfering with the mechanism of action of at least one agent that causes food allergies, preferably celiac disease. Preferably, the microorganisms selected in step a) belong to potentially probiotic genera or species.

Another aspect of the invention relates to microorganisms capable of hydrolyzing, inactivating or interfering with the mechanism of action of at least one agent that causes food allergies and which modulates the immune response. These microorganisms may be preferably used for the treatment or prevention of food allergies, inflammatory bowel diseases, imbalances in the intestinal ecosystem (dysbiosis), bacterial overgrowth, malabsorption syndrome, gastrointestinal infections and translocation of pathogenic microorganisms or their toxins, and changes of intestinal permeability. The invention also provides microorganisms useful for producing formulations which reduce risks and improve the health status of individuals suffering from or prone to suffering from disorders related to gluten intake through various mechanisms of action characterized in that they are microorganisms not genetically modified, isolated and selected from the intestinal microbiota of healthy individuals for their anti-inflammatory and regulatory properties.

Its multiple mechanisms of action include, for example, and without limiting the scope of the invention: (i) regulating the innate and adaptive immune response elicited by toxic and immunogenic gluten peptides; (ii) reducing the concentration of toxic epitopes in the lumen of the gut by transporting and hydrolyzing the gluten peptides; (iii) strengthening the defensive barrier function against bacteria or other pro-inflammatory agents and with virulence factors isolated from the gastrointestinal tract of celiac patients, and (iv) providing enzymatic activities, in addition to peptidases, which promote digestion and provide the nutrients to alleviate the typical malabsorption syndrome of these patients. This micro-organism can produce beneficial effects in addition to these by way of illustration and without limiting the scope of the invention, reducing the oxidative stress associated with inflammation, regulating intestinal permeability, favoring the colonization of beneficial Gram-positive bacteria with protective functions, regulating the function of antigen-presenting cells, inhibiting the interaction of toxic peptides with immunocompetent and epithelial cells of the host, interacting with metalloproteases, regulating the cell cycle and apoptosis, regulating cell growth and differentiation, and regulating neuroendocrine functions. (De Stefano et al., 2007). Lycopene, Guercetin and tyrosol prevent macrophage activation induced by gliadin and IFN-γ, Eur J Pharmacol 2, 566 (1-3): 192-9, Silano et al. Decapeptide from durum wheat prevents celiac peripheral blood lymphocytes from activation by gliadin peptides Pediatr Res. 61 (1): 67-71; Gross et al., 2007 Role of neuropeptides in inflammatory bowel disease Inflammation Bowel Dis. 918-32).

The microorganisms of the invention preferably belong to the genus Blfldobacterium. Microorganisms of this genus offer advantages in food formulations, novel foods, probiotics, symbiotics, supplements, nutraceuticals, and pharmaceutical formulations for the treatment or prevention of celiac disease and related disorders. Bifidobacteria have a special ability to colonize the intestinal tract of newborns, contributing significantly to the development of their defenses.

The microorganisms of the invention belong to the species B. longum. As an example, and without limiting the scope of the invention, the strain of the invention belonging to this species was isolated from the feces of healthy breast fed children and identified by sequencing the 16S rRNA gene (Example 6) and the tuf gene. The sequenced fragment (1437 bases) was amplified by PCR using primers 27f and 1401 and the sequencing primers 530f and U-968f were also used according to the procedures described by other authors (Satokari et al., 2001 Appl. Environ Microbiol 67, 504-513, Favier et al., 2002 Appl Appl Environ Microbiol 68, 219-226). By aligning the sequences obtained with 21 ΕΡ 2236598Β1 those found in the databases (GenBank), a maximum similarity was detected with the sequences equivalent to 14 different strains of the B. longum species and among them B. longum BG3 (accession number AY735403 .1). Part of the tuf gene (498 bp) was amplified and sequenced using the primers described by Ventura et al. (Analysis, characterization, and loci of the tuf genes in Lactobacillus and Bifidobacterium species and their direct application for species identification Env Environ Microbiol 2003, 69 (11): 6908-22). In this case, a maximum similarity was also detected with B. longum species and specifically with the sequence corresponding to strain B. longum ATCC 15707 (accession number AY372042.1) following the same procedure.

This demonstrates that B. longum exhibits optimal properties for regulating the immune system of the invention in a manner similar to other appropriately selected strains of this genus and species that can do so, providing the same beneficial effects in subjects with disorders related to gluten intake. The invention relates to a bacterial strain belonging to the species B longum and which has been deposited at the Spanish Collection of Types of Cultures (Spanish Collection of Crop Cultures - CECT), established in Burjassot (Valencia) on 20 December 2007, corresponding to the deposition number CECT 7347. This strain belongs to the species B. longum according to the sequence homology of the 16S rRNA gene with others normally available in the databases (GenBank), as described in example 6; as well as with the homology of the tuf gene with that of other strains of this species. The latter is an example of a strain of the genus Bifidobacterium 22 ΕΡ 2236598Β1 which has properties that allow its use in pharmaceutical formulations or medicaments, foods (novel or functional) or nutritional or dietary supplements. The strain of the invention may be combined with other microorganisms and bioactive compounds to enhance its protective and metabolic properties through synergistic or complementary actions such as increased total synthesis of regulatory cytokines and their types, increased inhibitory capacity against bacteria pathogenic and intestinal barrier function, and increased contribution of peptidases and other enzymes that favor digestion by increasing their total concentration or increasing their type and specificity.

Thus, another aspect of the invention comprises combining bifidobacteria with other microorganisms or bioactive compounds in a complementary and / or synergistic manner, favoring immunoregulatory responses and degradation of toxic and immunogenic gluten peptides. As an example of microorganisms, strain B. longum ATCC 15707 may enhance the immunomodulatory effect of the strain of the invention because of its high ability to induce IL-10 synthesis as shown in Table 1 (Example 1); this action may also complement the induction of TGF-β produced only by the second strain (Table 1, Example 1). At the same time, the strain Lactococcus lactis NCD0712 can complement and enhance the degradation of gluten peptides due to bifidobacteria and thereby reduce the concentration of toxic epitopes of gluten and intestinal and extraintestinal damage due to the fact that in addition to peptidases intracellular, extracellular proteolytic activity. The enhancement of proteolytic activity is demonstrated in Example 2 and Figure 1 where it is possible to appreciate a near complete disappearance of the protein bands after incubation of the digested samples of gliadin with cell suspensions of the strain of the invention and Lactococcus lactis NCD0712 ( Figure 1, panel B, lanes 3 and 4). This hydrolysis was superior to that obtained using only the strain of the invention (Figure 1, panel A, lanes 3 and 4).

Another aspect of the disclosure relates to the use of the strain of the invention, as well as its non-viable counterparts inactivated by different processes (freezing, heating, radiation, etc.) for immunomodulating purposes.

Non-viable microorganisms of the invention inactivated by different processes (freezing, heating, radiation, etc.) continue to be useful for therapeutic or preventive purposes and also form part of the present invention. The immunomodulatory effects of bifidobacteria are produced, at least in part, by structural constituents (DNA, cell wall components, etc.). In the present study, Bifidobacteria were found to be responsible for the pathophysiology of Bifidobacteria, which is characterized by the presence of Bifidobacteria and their pathogenicity. Microbiol., 38: 165-72). Thus, Example 3 and Table 3 demonstrate that the cell suspensions of the strain of the invention, inactivated by freeze-thaw cycles, can modulate the proinflammatory response triggered by gliadins when co-incubated with peripheral blood mononuclear cells, thereby reducing the synthesis of the inflammatory pro-inflammatory cytokines (eg IFN-γ) and enhancing the synthesis of regulatory cytokines (eg IL-10).

Also, the bioactive compounds derived from the microorganism of the invention such as structural compounds, those resulting from metabolism, the molecules secreted by any of the microorganisms or strains of the invention, obtained by well known techniques, form a part of the present invention and may also be used to modulate immune responses. For example, physicochemical and biotechnological techniques including, among others, centrifugation, filtration, lyophilization, precipitation, sonication, mechanical and chemical cell disruption, extraction of compounds based on cultures with enzymes and / or chemical agents, separation through chromatographic techniques, cloning of genes encoding said compounds and their overexpression. Example 1 and Table 1 demonstrate that the structural components that form part of the cell envelope of bifidobacteria are responsible at least in part for the induction and production of regulatory cytokines (IL-10 and TGF-β). In Example 3 and Table 3 in which the digested gliadin samples were co-incubated with non-viable cell suspensions of the strain of the invention it is also shown that the structural components of these cells are able to reduce the proinflammatory responses performed by gliadins and to increase to the synthesis of regulatory cytokines (IL-10). Likewise, structural components, metabolites and substances secreted by the strain of the invention exert an inhibitory effect on the growth of potentially pathogenic bacteria isolated from patients with celiac disease, as demonstrated in Example 4 and Table 4. In said example, inhibitory effect of the cell cultures of this strain has been evaluated using the double layer technique in which both cells as well as the metabolites and secreted products are brought into contact with the pathogenic microorganism so that the inhibitory effects may be due to the synergistic action of all these components. The inhibitory effect of the metabolites and compounds secreted by the bifidobacteria into the culture medium was also evaluated using as an inhibitor the supernatants from previously freeze-dried cell cultures. Thus, it has been shown that the compounds released into the culture medium provide an inhibitory effect against pathogenic potentials isolated from patients with celiac disease (Table 5).

In a particular embodiment of the present invention the microorganisms of the invention, the composition of the invention, the supernatant of the invention, the extract of the invention, the composition of the pharmaceutical or nutritional invention, the strain of the invention, are characterized by being able to regulate or modulate the innate and adaptive immune response elicited by gluten-damaging peptides or other food allergies. The strain of the invention was selected for its immunomodulatory properties capable of regulating Th1-type proinflammatory responses characteristic of celiac disease and related diseases (Down Syndrome, type 1 diabetes mellitus, dermatitis herpetiformis, myopathy, multiple sclerosis, arthritis, autism, schizophrenia, depression, lymphomas, and ataxia) as well as Th2-type allergic reactions that can be elicited as a result of ingestion of wheat and other cereal proteins. The strains are characterized by their ability to induce a low production of the cytokine Th1 IFN-γ (e.g. < 100 μg / ml) and the pro-inflammatory cytokine IL-1 (e.g. < 150 μg / ml) and induce a (eg > 800 pm / ml) and TGF-β (e.g. > 50 pmol / ml) by peripheral blood mononuclear cells (PBMCs). The cytokine profile induced by these bifidobacteria is not a feature common to all human lactic acid bifidobacteria and intestinal bacteria (Example 1, Table 1) and makes it especially ideal for modulating the abnormal immune response that wheat proteins produce in predisposed subjects and in patients with active celiac disease. Detection of these immunomodulatory effects when using cell suspensions of the selected strain as the stimulus in these tests (Example 1 and Table 1) indicates that the structural components that form part of the cellular envelope of this bifidobacteria are responsible, at least in part, for the induction of the production of the regulatory cytokine (IL-10 and TGF-β) that can reduce the toxic and immunogenic effects of gluten peptides. Also, in Example 3 and Table 3 it is shown that the use of non-viable cell suspensions of the strain of the invention (inactivated by freeze-thaw cycles) co-incubated with digested gliadin samples is able to reduce the synthesis of pro-inflammatory cytokines (for example INF-γ and IL-15) caused by gliadins and enhancing the synthesis of regulatory cytokines (for example INF IL-10). Therefore, it is demonstrated that the structural components of bifidobacteria cells can regulate the abnormal immune responses caused by gluten without it being strictly necessary to maintain the viability of the bacterium. The strain of the invention and the bioactive compound derived from the invention are characterized in that they are capable of strengthening the defensive barrier function against harmful bacteria, for example proinflammatory bacteria and virulence-like bacteria isolated from the gastrointestinal tract of patients of celiac disease.

The microorganisms of the invention are capable of inhibiting bacteria with pathogenic and toxic potential, isolated from the intestine of celiac disease patients (Example 4, Tables 4 and 5). These pathogens include, but are not limited to, strains of Escherichia coli species encoding pathogenicity factors (eg fimbriae) and belong to virulent phylogenetic groups (e.g., B2), strains of the genus Bacteroides and other genera producing metalloproteases that contribute to tissue injury, and hemolytic strains isolated from duodenal biopsies In Example 4 and Table 4 the inhibitory effect of total cell cultures of this strain is demonstrated against pathogens isolated from celiac disease patients using the double-layer technique , so that these effects may be due to structural components, metabolites and substances secreted by the strain of the invention. Table 5 also shows the inhibitory effect of cell-free supernatants from cultures of this strain which contain only the metabolites and compounds secreted by bifidobacteria to this medium. In both cases, the inhibitory effects obtained using the selected strain are superior to those obtained using other strains tested for comparative purposes. Thus, the microorganisms or the strain of the invention may contribute to restore the intestinal ecosystem and reduce the antigenic load of microbial origin which would favor the inflammatory process and increase the permeability of the epithelium. Likewise, the selected strain is capable of inhibiting the synthesis of proinflammatory cytokines (eg IFN-γ and TNF-Î ±) stimulated by the intestinal microbiota of celiac disease patients in peripheral blood mononuclear cells. For example, concentrations of IFN-γ of 90.8 and TNF-α and 1966.3 pmol / ml produced by PBMCs under stimulation with the microbiota of celiac disease patients may be reduced to 8.2 pmol / ml and 295.2, respectively, in the presence of the strain of the invention (Example 7). This strain is also capable of stimulating the synthesis of regulatory cytokines (IL-10), reduced at the same time by the intestinal microbiota of celiac disease patients. Thus, for example, IL-10 values of 49.3 pmol / ml induced by the microbiota of celiac disease patients can be increased to values of 107.5 pmol / ml by stimulation of the Bifidobacterium IATA-H1 strain. Through this immunomodulatory mechanism the selected strain can contribute to restore intestinal balance and avoid the over stimulation of the immune system caused by the harmful microbiota that, together with that caused by gluten, can create a vicious circle that perpetuates inflammation.

Also, the microorganisms of the invention are characterized by their ability to carry the gluten peptides that result from gastrointestinal digestion, thereby reducing the concentration of injurious epitopes. 29 ΕΡ 2236598Β1

Specifically, the strain of the invention has the ability to recover toxic peptides derived from the gastrointestinal digestion of gliadins, from products that result from gastric digestion through the action of pepsin (P), as well as from intestinal digestion through the action of trypsin (T) and pancreatin (X) (Example 2, Figure 1). Incubation of gliadins in the presence of viable bacteria of the selected strains reduces their concentration and the presence of toxic epitopes determined using the R5 antibody by sandwich ELISA and thus their possible harmful effects on the gut and the extraintestinal level. For example, the concentration of toxic epitopes of a sample of glidin digested with pepsin, trypsin and pancreatin (as indicated in Example 2) of 2349 ppm gluten determined by sandwich ELISA was reduced to 169 ppm gluten after incubation with strain of the invention. Likewise, incubation of the strain of the invention with gastrointestinal and dialysed digested gliadin with a membrane size exclusion of less than 15 kDa, and its subsequent analysis by reverse phase HPLC made evident the ability of this bifidobacterium to reduce at least in 10% the concentration of fraction # 2, which was the main fraction of digested gliadins and that containing immunogenic peptides identified by mass spectrometry, a property that the rest of the strains tested did not show (Example 8, Figure 2). The biological effects on the intestinal epithelium of this property of the B. longum IATA-ES1 strain were demonstrated after incubation of the different digested gliadin samples in Caco-2 cell cultures as described in Example 8. This example demonstrates that the strain of the invention may thereby increasing the viability of the 30 Ρ 2236598Β1 epithelial cells, unlike the rest of the strains tested (Example 8, Figure 3). In addition, co-incubation of the strain of the invention with the digested gliadin sample decreased the effect of the latter on the synthesis of proinflammatory cytokines such as TNF-alpha and expression of the responsible NkB factors for expression of proinflammatory genes (Example 8, Figure 4). The ability of the strain of the invention to capture oligopeptides derived from the digestion of gliadin is reproduced under intestinal conditions and in the presence of salts of bile. This capacity is potentiated by co-incubation with Lactococcus lactis NCD0712 which, although it can not colonize the large intestine, can act as a coadjuvant in the early stages of gluten hydrolysis ingested through the action of its protease anchored to the wall and other peptidases (Example 2, figure 1). The enhancement of proteolytic activity through the action of Lactococcus lactis NCD0712 is demonstrated in Figure 1 in which a near complete disappearance of the protein bands can be appreciated after incubation of the digested samples of gliadin with cell suspensions of the strain of the invention and Lactococcus lactis NCD0712 (Figure 1, panel B, lanes 3 and 4). This hydrolysis was superior to that obtained using only the strain of the invention (Figure 1, panel A, lanes 3 and 4). The activity of bifidobacteria in gluten can also be potentiated through their co-incubation with proteases and peptidases of Lactococcus lactis NCD0712 in the form of extracts. The strain of the invention may modulate the abnormal immune response elicited by the interaction of the toxic peptides of gluten with the immunocompetent cells of the individual not only by metabolizing the peptides that act as damaging antigens but also by immunoregulatory mechanisms (Example 3, Table 3). The viable or inactivated bacterial suspensions of the selected strains co-incubated with PBMCs in the presence of gastrointestinal digested gliadin samples are able to counteract the proinflammatory effect of these proteins at different stages of digestion by gastric (P) pepsin and intestinal trypsin (T) and pancreatin (X). This strain is capable of inducing a reduction in the production of the proinflammatory cytokines IFN-γ, IL-1, IL-8 and IL15 responsible for the innate and adaptive immune response, as well as enhancing the synthesis of the IL-10 regulatory cytokine (Example 3 , Table 3). These effects are due, at least in part, to inhibition of the different nuclear factor (NF) kB, p50, p65 (RelA), c-Rel and King B subunits determined by an ELISA assay (Trans AM NFkB, Active Motive, Belgium) . Inhibition of this transcription factor implies the inhibition of expression of a large number of proinflammatory genes that represent a key control point of inflammatory processes and specifically those occurring in celiac disease (Jelínková et al., 2004). Gliadin stimulates human monocytes to production IL-8 and TNF-alpha through a mechanism involving NF-kB FEBS Lett. 571 (1-3): 81-5).

The microorganisms of the invention and the bioactive compounds derived from the invention are characterized in that they are capable of hydrolyzing gluten peptides through their peptidase activity, reducing the concentration of detrimental epitopes. As an example, the strain of the invention has broad specificity and specificity peptidases for proline-containing substrates which are very abundant in gliadins and 32 Ρ 2236598Β1 limit their hydrolysis by conventional enzymes (Example 2, Table 2). Specifically, this strain is one of those with the highest iminopeptidase activity (> 300 U / mg protein); (> 8 U / mg protein), X-prolyl-dipeptidyl-peptidase (> 15 U / mg protein), prolidase and prolinase activity (> 15 U / mg protein ). It also possesses tripeptidase activity (> 130 U / mg protein against the Gleu-Gly-Gly substrate) and leucil-aminopeptidase activity (> 70 U / mg protein). The activity of this strain against proline-containing substrates such as Pro -pNA activity is higher than that detected in other lactic acid bacteria and especially the proportion of Pro-pNA / Leu-pNA activity (Di Cagno et al., 2004). (1): 80-93 (1999), pp. 253-223, and the author of this article, and the author of this paper, ), which favors the specific hydrolysis of gluten peptides with a high proline content.

Thus, the strain of the invention and the bioactive compounds derived from the invention have additional metabolic activity to that of the peptidases (for example: phosphatase, esterase, lipase, galactosidase, glucosidase and N-acetylglucosaminidase) which aid in the digestion of nutrients which are ingested with diet, and improve the malabsorption syndrome and malnutrition characteristics of patients with celiac disease. 33 ΕΡ 2236598Β1

Finally, another particular embodiment is the use of the microorganism of the present invention and the bioactive compounds derived from the invention in combination with other microorganisms in the production of formulations to reduce risks and improve the health status of patients with diseases related to ingestion gluten, or other food allergies.

Formulations prepared using the strain of the invention can be industrially developed giving rise to, inter alia and without limiting the scope of the invention, different forms of presentation to the consumer: foods (functional, conventional and novel), supplements, nutraceuticals, pharmaceutical compositions, probiotics and / or symbiotic, or novel foods. The strain of the invention has the ability to adhere to mucin (1-4%) and is resistant to acidic pH of the stomach (2.0, 2.5 and 3.0) and to high concentrations of bile salts (0.5 , 1.0, 2.0 and 3.0%) present in the small intestine, which are the main biological barriers limiting the survival of probiotics when they pass through the intestinal tract as well as during the food fermentation processes and their period (Example 5, Table 6). For example, this strain maintains a viability and growth capacity of 56-86% after 90 min of incubation at pH 2.0-2.5. Therefore, this strain has higher probabilities of survival and of being functional than other isolates and than some commercially available probiotics, as can be seen from Table 6. This strain also resists gastrointestinal transit in vivo. After administration in the form of fermented milk 34 Ρ 2236598Β1 for 4 weeks at doses of 107-108 cfu / ml twice daily is recovered in the faeces and its concentration in relation to the initial concentration (without ingestion of probiotic products) is increased in at least least 1 logarithmic unit. The selected bifidobacteria grow and remain viable in various foods and beverages, constituting ideal vehicles for their incorporation. For example, these are capable of fermenting and coagulating milk, these can be used for the preparation of fermented milks and other milk derivatives. These also resist technological treatments for the production and preservation of foods, supplements and pharmaceutical formulations, such as for example lyophilization and refrigeration temperatures, guaranteeing their industrial exploitation.

One particular aspect of the present invention comprises the use of the strain of the invention or bioactive compounds derived from the invention in the preparation of formulations in the form of foods.

In this way, the microorganisms of the invention may form part of a food formulated to provide, beyond its normal nutritional value, a beneficial accomplishment in reducing risks and improving the health status of patients with gluten-related diseases.

Another particular aspect of the present invention comprises the use of the strain of the invention or the bioactive compounds of the invention in the production of preparations in the form of nutraceuticals defined as natural bioactive substances presented in a non-food matrix which in this case should have beneficial effects in patients with diseases related to gluten intake, reducing their risks and improving their health status.

In the case of the use of the microorganisms, the strain of the invention or compounds of the invention derived therefrom to obtain a dietary or food supplement should include in its composition the microorganism or its bioactive compounds derived from it to supplement the diet for health purposes and in this specific case, with the aim of producing beneficial effects in patients with gluten-related diseases, reducing their risks and improving their health.

Another particular aspect of the present invention comprises the use of the microorganisms of the invention, the strain of the invention or bioactive compounds of the invention in the production of pharmaceutical preparations. In this way, they will be used in the preparation of biologically captive compositions capable of being used as medicaments producing a beneficial effect in reducing risks and improving the health status of patients with diseases related to gluten intake.

In another particular embodiment of the invention, the strain of the invention or bioactive compounds of the invention should be used in the production of probiotics and / or symbiotics (combinations of probiotics and prebiotics), wherein the microorganisms are incorporated, for example live or lyophilized, into adequate amounts and under conditions which enable them to effect their beneficial or therapeutic effect in individuals with food allergies, preferably those related to the ingestion of gluten, thereby reducing their risks and improving their health status. 36 ΕΡ 2236598Β1

A particular end goal of the present invention comprises the use of the strain of the invention or bioactive compounds of the invention in the preparation of novel foods. It is understood that novel foods mean any food or ingredient which has not been normally used in the past for human consumption in the European Union before 15 May 1997, which would produce beneficial effects in patients suffering from gluten-related diseases, their risks and improving their state of health.

EXAMPLES OF EMBODIMENTS OF THE INVENTION EXAMPLE 1. PROCESS FOR SELECTING BIFIDOBACTERIUM GENDER STRENGTHS ACCORDING TO ITS CAPACITY TO MODIFY CYTOKINE PRODUCTION IN PERIFERIC BLOOD MONONUCLEAR CELLS (PBMCs) 1. Preparation of cultures and supernatants of blfidobactles and other bacteria intestinal diseases.

The strains were inoculated in 10 ml of MRS culture medium (Scharlau Chemie SA, Barcelona, Spain) containing 0.05% cysteine (MRS-C) at 1% with a 24 h culture and incubated for 22 h at 37øC in anaerobiosis. (AnaeroGen, Oxoid, Basingstoke, UK). Cells were harvested by centrifugation (6000 g, 15 min), washed twice in PBS (10 mM sodium phosphate, 130 mM sodium chloride, pH 7.4), and resuspended in PBS containing 20% glycerol. Fractions of these suspensions were frozen using liquid nitrogen and stored at -80Â °. The number of viable cells after the freeze-thaw cycle was determined by counting on MRSC plates after incubation for 48 h. 37 ΕΡ2236598Β1 Viability was above 90% in all cases. Each fraction was used during a single test. In order to evaluate the effects of dead bacteria, some of the fractions were inactivated by cold (3 cycles of freezing at -20 ° C and thawing) and inactivated by heat (30 min at 80 ° C). The pH values of the obtained supernatants were adjusted to 7.2 using NaOH and sterilized by filtration (0.22 mm pore size, Millipore, Bedford, MA) to eliminate the possible presence of viable cells. Fractions of cell-free supernatants were stored at -80øC until further use.

2. Isolation and stimulation of PBMC

PBMC were isolated from the peripheral blood of 4 healthy volunteers (mean age 30 years, range 24-40) in heparin tubes. PBMCs were isolated by Ficoll gradient centrifugation (Amersham Biosciences, Piscataway, NJ). The cells were washed with RPMI 1640 medium (Cambrex, New York, USA) and adjusted to a density of 1 x 106 cells / ml in RPMI 1640 medium containing additionally 10% fetal bovine serum (Gibco, Barcelona, Spain), 2 mM L-glutamine, 100æg / ml streptomycin and 100 U / ml penicillin (Sigma). PBMCs were incubated in 24-well flat-bottomed polystyrene plates (Corning, Madrid, Spain) in the presence or absence of stimulating agents at 37øC, 5% CO 2 for 24 h. Suspensions of live and dead bacteria were used as a stimulus at 1 x 10 6 CFU / ml, and volumes of supernatant 150 μl. As a positive control, purified lipopolysaccharides (LPS) from E. coli 0111: B4 (Sigma, St. Louis, MO) at a concentration of 1 pg / ml were used. As a negative control, the production of cytokines in unstimulated PBMC was tested. Each type of stimulus was tested in duplicate in each experiment. Culture supernatants were collected by centrifugation, fractionated and stored in fractions at -20 ° C until detection of cytokine. 3. Determination of cytokine

The cytokine concentrations (IL-1, IFN-γ, IL-10, and TGF-β) of the supernatants were measured using ELISA Bioscience kits (BD Biosciences, San Diego, CA) following the manufacturer's instructions.

Table 1. Immunomodulatory properties of bifidobacteria and other intestinal lactic bacteria. Effect on the production of cytokines by the PBMC of viable bacteria.

Cytokine production (μg / mL) IL-1 IFN-γ IL-10 TGF-β RPMI ND 9.0 ± 1.0 58.0 ± 3.0 ND LPS ND 12.0 ± 0.5 399.0 ± 8.0 ND + S 103.0 ± 37.0 10.1 ± 1.0 2459.0 ± 28.0 236.0 ± 119.0 2A 2 255.0 ± 1.0 13.0 ± 2.0 699.0 ± 396.0 ND 3ATCC15707 - 66.1 ± 23.9 4098.4 ± 1551.7 - 4BIR-324 ND 11.0 ± 5.0 469.0 ± 15.0 ND 5W11 - 160.4 ± 6.8 486.0 ± 236.4 - 6BB536 - 143.7 ± 18.3 1390.0 ± 268.8 - 7lmiv 233.0 ± 99.0 27.0 ± 15.0 166.0 ± 53.5 ND ND, unrecognized unreacted 1Estape of the invention (IATA-ES1), 2Bifidobacterium IATA-A2, 3Bifidobacterium longum ATCC15707, 4Bifidobacterium BIR-324, sBifidobacterium longum Wll, 6Bifidobacterium longum BB536,? Lactobacillus reuteri LM1V. 39 ΕΡ 2236598Β1

EXAMPLE 2. A PROCESS FOR SELECTING HYDROLYSIS CAPABLE BIFIDOBACTERES AND TRANSPORTING GLUTEN PEPTIDES REDUCING THEIR TOXICITY THEREFORE The ability of the strains to hydrolyze gluten-derived proteins and peptides was measured by quantifying the broad spectrum activity and specific intracellular peptidases to hydrolyze proline-containing peptide sequences present in the peptides responsible for the immunological and toxic responses of gluten. Cells from 16-18 hours bacterial cultures cultured in MRSC were harvested by centrifugation (9000 xg for 10 minutes at 4øC) washed twice in 50 mM Tris buffer pH 7 and resuspended in the same buffer 10 times concentrated to the volume of the initial culture. Cells were mechanically busted using a Bead-Beater (Biospec Products, USA) by adding 2 volumes of glass beads to each cell volume and applying 2 pulses of 1.5 minutes. The supernatant obtained after centrifugation (8000 g, 10 min) to remove insoluble fragments and cells was used as an enzymatic extract for the activity tests. The substrates tested were as follows: Leu-paranitroanilide (-pNA) to detect broad specificity aminopeptidases, Leu-Leu-Gly to detect aminopeptidases and wide specificity tripeptidases, Suc-Ala-Pro-pNA to detect prolyl endopeptidases, Pro -AMC to detect iminopeptidases, Gly-Pro-AMC to detect X-prolyl-dipeptidylpeptidases; Val-Pro to detect prolidases, and Pro-Gly to detect proinases. In the case of substrates derived from paranitroanilide, the reaction mixture consisted of 200 μΐ of 50 mM phosphate buffer, pH 7.2, containing 0.5 mM of substrate and 50 μΐ of enzyme extract. Reaction mixture 40 Ρ 2236598Β1 was incubated at 37øC for a maximum of 30 min. Hydrolysis of the substrate and the release of paranitroaniline was monitored at 419 nm in a spectrophotometer (550 Microplate Reader, Bio-Rad, Hercules, CA, USA). The hydrolysis of the peptides was determined by the L-amino acid oxidase assay (Hejgaard, 1978, Rapad assay to detect peptidases in column effluent fractions using L-amino acid oxidase, Analytical Biochem 90: 835-839). 100 μl of the reaction buffer containing a concentration of 0.05 mM was incubated with 50 μl of enzyme extract for 20 min. After this time 100 μΐ of the L-amino acid oxidase reagent was added and, after 5 minutes of incubation, the absorbance was measured at 530 nm. Protein concentration was measured using the Bradford method with the commercial BioRad kit (Hercules, CA, USA).

One unit of activity was defined as the amount of enzyme capable of hydrolyzing 1 pmole of substrate at 37 ° C for 1 minute. Activities were expressed as U / mg protein. The ability of the strains to carry and hydrolyze the peptides derived from the digestion of gliadins was determined by electrophoresis. In this case, the cell suspensions were adjusted to an optical density of 4 to 655 nm, equivalent to 109 cfu / ml, and incubated with three different gliadin hydrolysates at a final concentration of 300-600 pg / ml in PBS to which 0.2% glucose added. Gliadin hydrolysates (Sigma, St. Louis, MO) were obtained by stimulation of the gastrointestinal digestion process as follows: A). 100 g of gliadins were digested in one liter of 0.2 N HCl (pH: 1.8) with 2 g of purified pepsin at 37ø for 2 h (this hydrolyzate will be referred to as G-P). B) The resulting digestion was digested with trypsin by the addition of 2 g of trypsin Ρ36 2236598Β1 after adjusting the pH to 8 using 2N NaOH (this hydrolyzate will be referred to as G-P + T CC). The digested double sample was then treated with 2 g of pancreatin and stirred for 2 hours at pH 8 (this hydrolyzate will be referred to as G-P + T + X). After each stage of digestion it was centrifuged at 10,000 g for 10 min, and the supernatant was stored for further testing at -20 ° C. The digested samples were inactivated by incubation at 100 ° C for 30 minutes. Bacterial suspensions were incubated in the presence of the three gliadin hydrolysates (A, B and C) for 6 hours at 37øC under anaerobic conditions. Changes in viability during incubations were determined using the LIVE / DEAD BacLight Kit commercial system (Molecular Probes, Leiden, The Netherlands) for microscopy following the manufacturer's instructions. Count of live (green) and dead (red) bacteria was performed on an Olympus BX 51 epifluorescence microscope (Tóguio, Japan). In all cases, minimal losses of viability (0.0-11.5%) were detected after incubation. After the incubation period it was centrifuged to remove insoluble cells and proteins and the supernatants were sterilized by filtration (0.22 mm pore size, Millipore, Bedford, MA) to eliminate the possible presence of viable cells. The ability of the strains to carry and use the different gliadin hydrolysates was determined by assessing the disappearance of bands on conventional 15% polyacrylamide gels and Tris-Tricine gels (Ready gels 10-20% linear gradient, 4% packaging, Bio-Rad, Barcelona, Spain) for peptide separation. Proteins were visualized by staining with Coomassie Brilliant Blue R-250. The reduction in toxic epitopes resulting from the transport and digestion of gliadins through the action of the selected bacteria was evaluated using a 42 ΕΡ2236598Β1 R5 sandwich ELISA assay (Centro Nacional de Biotecnologia, Madrid).

Table 2. Activity of peptidase from strains of bifidobacteria and lactobacilli of intestinal origin against synthetic substrates

Pro-Gly Leu-Gly-Gly Pro-pNA Leu-pNA Suc-Ala-Pro-pNA Gly-PropNA 1ES1 18.1 ± 3.5 15.7 ± 3 , 8 132.3 ± 7.2 480.2 ± 91.7 80.0 ± 8.9 ± 8.5 ± 1.9 17.1 ± 0.0 ± A2 27.1 ± 14.0 27.1 ± 07.5 326.1 ± 15, 140.5 ± 6.4 176.5 ± 3.5 18.5 ± 4.8 60.43 ± 11.0 15707 27.4 ± 2.0 37.9 ± 0.8 - - 5.1 ± 0.5 0.02 ± 0.6 0.2 ± 0.5 4 Lm VI 9.34 ± 0.7 10.3 ± 0.6 54.6 ± 2.7 7.8 ± 3.7 61.8 ± 3.4 8.0 ± 3.5 15.2 ± 3.2 * Mean ± SD, not evaluated 1Style of the invention (IATA-ES1), 2Bifidobacterium IATA-A2, 3Bifidobacterium longum ATCC 15707, 4Lactobacillus reuteri LM1V. EXAMPLE 3. REGULATION OF THE IMMUNOLOGICAL RESPONSE CAUSED BY GLADIADINES IN IMMUNOCOMPETENT CELLS THROUGH THEIR CO-INCUBATION WITH BIFIDOBACTERIUM GENUS SELECTED BY THEIR IMMUNOMODULATING PROPERTIES.

Suspensions of the selected strain (10δ-109 CFU / ml), as well as others included for comparative purposes, were incubated with the different gliadin hydrolysates (P, P + T and P + T + P), obtained as described in example 3 , and the PBMCs at a concentration of 106 cfc / ml for 24 h. As a control stimulus the different gliadin hydrolysates were used without bacteria and E. coli 0111: B4 LPS. Basal cytokine production was also detected without adding any stimulus. The procedure for isolating PBMCs, stimulating and detecting cytokines was described in example 1.

Table 3. Production of cytokines by PBMCs stimulated simultaneously by gliadins digested in gastrointestinal conditions and in the presence of infeasible cultures of bifidobacteria and other lactic bacteria with intestinal origin.

Cytokine production (pm / ml) IL-1 IL-8 Stimulus IL-8 IL-10 IL-10 IL-15 RPMI ND 173 + 17 9 + 1 58 + 3 ND LPS ND 2295 + 83 12 + 0.5 399 + 8 175 + 17 GP 61 ± 2 4570 + 484 4267 299 + 149 34 + 1 GP + T 169622 149 + 83 13 6 + 16 37 + 3 5375 + 13 G-P + T + X 7 5 ± 1 5264 + 122 36 + 9 ES2 / G-P + T 48 ± 4 4928 + 288 8, 2 + 5 935 + 426 27 + 10 ES1 / GP + GP + T + X ND 5151 + 146 2 + 0, 5 1054 + 477 ND A 2 / GP 109 + 215 4480 + 23 6, 9 + 2 395 + 216 29 + 8 A2 / G-P + T 129 + 4 2848+ 45 43 + 22 1002 + 615 46 + 13 A2 / G-P + T + X 129 + 1 5099 + 30 29 + 4 1314 + 724 134 + 57 3LM1V / GP ND 5152 + 119 62 + 31 721 + 16 33 + 4 LM1 V / G-P + T ND 5015 + 75 11 + 5 458 + 218 2 6 + 4 LM1 V / GP + T + X ND 5189 + 98 -82 + 35 457 + 257 6 + 1

ND, not detected 1Abstract of the invention (IATA-ES1) 2Bifidobacterium IATA-A2 3Lactobacillus reuteri LM1V 44 ΕΡ2236598Β1 EXAMPLE 4. CAPACITY OF BIFIDOBACTERIA SELECTED TO INHIBIT THE GROWTH OF ISOLATES OF INTESTINAL MIROBIOTA OF CELL PHEASED CELIAC DISEASES. The antimicrobial activity of strains of the genus Bifidobacterium previously selected according to their immunomodulatory properties following the procedure described in Example 1 was determined by two methods: (i) the double layer technique and (ii) the agar diffusion technique . The antibacterial activity of each strain was evaluated globally through the double layer technique using as indicator microorganisms, bacteria isolated from the intestinal microbiota of patients with celiac disease and with pathogenic potential. The bifidobacteria were plated on MRS-C plates in rows of about 2 cm and incubated under optimum conditions for 16 h and their subsequent development was inhibited using chloroform. The indicator microorganism was inoculated at a concentration of 104-105 CFU / ml in 10 ml of appropriate semi-solid agar, poured onto the agar layer of the protective microorganism and incubated at 37øC under anaerobic conditions. After 24 h the inhibition halos were measured around the culture lines of the bifidobacteria.

In order to evaluate the antibacterial activity due to the secretion of compounds of protein nature, the agar diffusion technique was used. 10 ml of 1% MRS-C growth medium was inoculated with a 24 h culture of each of the bifidobacteria and incubated for 16 h at 37 ° C. Supernatants were obtained by centrifugation (12,000 g, 15 min, 4 ° C) and concentrated by lyophilization. The lyophilized samples were resuspended in 1 ml of 50 mM phosphate buffer pH 6.5, neutralized with NaOH until a pH of 6.5 was reached to eliminate the effects of the organic acids produced by fermentation and sterilized by filtration. These samples constituted crude extracts in which the possible activity of antibacterial proteins produced by bifidobacteria was determined. The indicator microorganism was inoculated at a concentration of 104-105 cells / ml in 10 ml of semisolid agar and poured onto a solid layer of agar of the same medium. After solidification, the 5 mm wells were drilled, to which 40 μΐ of the cell-free and neutralized extract of each of the bifidobacteria were added. It was allowed to diffuse for 4 h at 4 ° C and was subsequently incubated under optimum conditions for the indicator or pathogenic microorganism. After incubation the inhibition halos around the well were measured.

Table 4. Inhibition of potentially pathogenic bacteria isolated cultures from patients of bifidobacteria. celiac by Strains Inhibition by (cm) Blfidobacterium spp IATA-A2 IATA-ESI Bacteroides CAQ4 0.4 1.4 Bacteroides vulgatus 0.5 1.3 Clostridium difficile 0.9 1.6 E. coli CBE9 1.1 1.9 BC-BP1 0.5 1.2 BC-B01 0.7 1.0 BC-BU1 2, 3 2, 0 BC-BU3 1.0 1.3 46 ΕΡ 2236598Β1

Table 5. Inhibition of potentially pathogenic bacteria isolated from culture supernatants of bifidobacteria from celiac patients.

Indicator strain Inhibition by (cm) Bifidobacterivaa spp. IATA-A2 IATA-ES1 Bac CAQ4 0.40 0.45 Ent CBE9 0.95 1.15

EXAMPLE 5. EVALUATION OF RESISTANCE OF BIFIDOBACTERIA TO THE CONDITIONS OF GASTROINTESTINAL STRESSE The resistance of the isolated bifidobacteria to the acidic conditions of the gastric juices, which are the first biological barrier limiting the viability of probiotics after ingestion, was confirmed for each of the strains recovered. To do this, the cell suspensions of each strain were prepared (108 cells / ml) in PBS containing 3 g / l pepsin (Sigma, St. Louis, MO) and adjusted to pH 2 with HCl and incubated at 37 ° C for a total of 120 min. At different times (0, 90 and 120 min), including mean gastric emptying time (90 min), aliquots were taken to determine viability by counts on MRS-C agar plates. Subsequently, the tolerance of strains resistant to acid pH to other stress conditions such as bile, NaCl and elevated temperatures was studied. To know the tolerance of the studied strains to bile tolerance, their ability to grow was evaluated in MRS-C, to which several concentrations (0.5-1.5%) of Ox-gall (Sigma, St. Louis , MO). 200 ml aliquots of each medium, inoculated at 1% with 47 Ρ 2236598 culturas 24 h cultures, were loaded into multiwell wells and incubated at 37øC. Growth was monitored using absorbance measurements at 655 nm on a 550 Microplate Reader spectrophotometer (BioRad, Hercules, CA).

Table 6. Effect of gastric conditions on growth capacity and viability of Bifidobacterium strains

Viability Growth capacity * <%) PH pH 3 2.5 2 Strains 3 2.5 2 Control Log cfu / ml t%) Log cfu / ml t%) log Cfu / ml t%) IATA-ES1 99.4 ± 4.2 86 ± 2 ± 13.0 57 ± 2.3 9 ± 3 ± 0.0 9.3 ± 0.0 99.778 ± 0.086, 0 56.1 1 BIR 324 89.4 ± 2.3 73.1 ± 1.06 61.6 ± 1.7 9.1 ± 0.0 8.8 ± 0.0 96.9 ± 8.8 ± 0, 2 96.8 ± 5.7 63 ± 6 Bion 3 98.4 ± 1.9 9 78.4 ± 1.7 7 11.6 ± 3.09 ± 0.0 7.3 ± 0 , 0 81.2 4.8 ± 0.1 52.8 NCIMB 8809 1.8 ± 0.5 8 ± 1 ± 0.0 Fe viability expressed as the percen Kit system (Molecular Probes) in s 100%. fCapacity of growth expressed and expressed as a percentage of being 100%. Mean standard deviations of the results of the assay of the results of the assay were not detected in the cell counts obtained with the suspension in three t detectable samples of PBS with a pH of 7.2 , counting the number of cells in the dependent cell, and the L1 that was 4 in the DBS cell, which is nd, n VE / DEAD Ba considered as cell counts, was considered as a determinant of the MRSC serotype 48 ΕΡ 2236598Β1

Table 7. Tolerance of Bifidobacterium strains to the presence of bile salts

Strains Capacity of relative growth * (%) Oxgall concentration (%) Control 0, 5 1.0 2.0 3.0 IATA-ES1 100 88.22 ± 0.70 82.65 ± 1.60 69.95 ± 0 , 33 67.28 ± 1.31 A2 100 73.03 ± 0.98 64.12 ± 1.79 60.96 ± 0.59 38.21 ± 1.44 BIR 324 100 62.68 ± 3.55 43 , 99 ± 1.64 38.29 ± 0.32 37.06 ± 0.48 NCIMB 8809 100 45.07 ± 8.20 23.88 ± 7.56 3.94 ± 1.05 0.70 ± 1, 00 Bion 3 100 30.17 ± 3.2 23.30 ± 0.0 - - * Data expressed as a percentage of the growth rate (h -1) obtained in the absence of bile, which was considered to be 100%. The mean standard deviations of the results obtained in three independent experiments.

EXAMPLE 6. ISOLATION AND IDENTIFICATION OF SELECTED BIFIDOBACTERIAS

Strains of the genus Bifidobacterium were isolated from feces from healthy, breast fed infants who had not ingested foods containing bifidobacteria for at least one month prior to analysis and who had not been given any antibiotic treatment. Samples were kept at 4 ° C and analyzed without more than two hours after collection. Two grams of each were diluted in 10 mM phosphate buffer containing a concentration of 130 mM NaCl (PBS) and homogenized in a Lab-Blender 400 digester (Seward Medical, London, UK) for 3 min, and then diluted with water peptone 0.1 ml aliquots of various decimal dilutions were inoculated onto MRS agar (from Man Rogosa and 49 ΕΡ 2236598Β1

Sharpe; Scharlau, Barcelona) containing 0.05% cysteine (Sigma, St. Louis, MO; MRS-C), and 80 mg / mL mupirocin. After 48 hours of incubation at 37øC under anaerobic conditions (AnaeroGen, Oxoid, UK) the isolated colonies were selected and their identity confirmed by a study of their morphology under Gram staining. The identity of the isolates was confirmed by gender specific PCR, according to the methodology described by Kaufman et al. (1997, Identification and quantification of Bifidobacterium species isolated from food with genus-specific 16S rRNA targeted probes by colony hybridization and PCR Appl Appl Environ Microbiol 63: 1268-1273) using primers (LM26 and LM3) that amplify a fragment of the 16S ribosomal RNA gene. Also, the 16S rRNA gene was sequenced from total DNA. The sequenced fragment was amplified using primers 27f and 1401r and purified using the commercial GFX ™ PCR system (Amershan, Bioscience, UK). For sequencing, primers 530f and U-968f were also used according to the procedures described by other authors (Johnson, 1994. Similarity analysis of rRNAs, In Methods for General and Molecular Bacteriology, Gerhard, P .; Murray, RGE; Wood, WA, Krieg, NR, Eds, American Society for Microbiology, Washington, DC, pp. 683-700, Satokari et al., 2001. Bifidobacterial Diversity in Human Feces Detected by Genus-Specific PCR and Denaturating Gradient Gel Electrophoresis Appl. Microbiol 67, 504-513; Favier et al., 2002 Molecular Monitoring of

Succession of Bacterial Communities in Human Neonates. Appl. Environ. Microbiol. 68, 219-22). Sequencing was performed using an ABI 3700 automatic DNA sequencer (Applied Biosystem, Foster City, CA). The search for the related sequences was performed in the GenBank database 50 ΕΡ 2236598Β1 using the BLAST algorithm (Altschul et al., 1990. Basic local alignment search tool, J. Mol Biol. 215, 403-410). EXAMPLE 7. EVALUATION OF CAPACITY OF BIFIDOBACTERIUM GENDER STRENGTHS TO REGULATE THE PRO-INFLAMMATORY RESPONSES CAUSED BY THE ALTERED INTESTINAL MICROBIOTY OF CELIAC INDIVIDUALS WITH ACTIVE DISEASE AND AFTER TREATMENT WITH A DIET FREE OF GLUTEN. 1. Preparation of intestinal cultures of bifidobacteria and fecal samples for evaluation.

Strains of the genus Blfldobacterium were inoculated into 10 ml of MRS growth medium (Scharlau Chemie SA, Barcelona, Spain) containing about 0.05% cysteine (MRS-C) at 1% with a 24 h culture and incubated for 22 h at 37 ° C in anaerobiosis. (AnaeroGen, Oxoid, Basingstoke, UK). Cells were harvested by centrifugation (6000 g, 15 min), washed twice in PBS (10 mM sodium phosphate, 130 mM sodium chloride, pH 7.4), and resuspended in PBS containing 20% glycerol . Aliquots of these suspensions were frozen using liquid nitrogen and stored at -80øC. The number of viable cells after the freeze-thaw cycle was determined by recounting MRSC plates after incubation for 48 h. Viability was greater than 90% in all cases. Each aliquot was used for a single test.

Feces of celiac patients with active disease (at diagnosis) and treated with a gluten-free diet for at least 2 years were diluted 1/10 in phosphate buffer, homogenized in a digester for 3-5 51 Ρ 2236598 minutos minutes and frozen at -20 ° C for use as a stimulation in peripheral blood mononuclear cells. Stool samples from healthy subjects were also taken as controls.

2. Isolation and stimulation of PBMC

Peripheral blood PBMC from 4 healthy volunteers (mean age 30 years, ranging from 24-40) were isolated in tubes with heparin. Isolation of PBMC was effected by Ficoll gradient centrifugation (Amersham Biosciences, Piscataway, NJ). Cells were washed in RPMI 1640 medium (Cambrex, New York, USA) and adjusted to a density of 1 x 106 cells / ml in RPMI 1640 medium also containing 10% fetal bovine serum (Gibco, Barcelona, Spain), 2 mM L-glutamine, 100 mg / ml streptomycin and 100 U / ml penicillin (Sigma). PBMCs were incubated in 24-well flat bottom polystyrene plates (Corning, Madrid, Spain) in the presence or absence of stimulation agents at 37øC, 5% CO 2 for 24 h. As a stimulus, fecal extracts from healthy subjects, patients with active and non-active celiac disease, were used in the presence or absence of 30 ml of live bacterial cell suspensions of 1 x 106 CFU / ml. As a positive control, purified lipopolysaccharide (LPS) from E. coli 0111: B4 (Sigma, St. Louis, MO) at a concentration of 1 pg / ml was used. As a negative control cytokine production in unstimulated PBMC was tested. Each stimulus type was tested in duplicate in each experiment. Culture supernatants were collected by centrifugation, fractionated and stored in aliquots at -20 ° C until detection of the cytokine. 3. Determination of Cytokines and Cell Activation Markers The concentration of cytokines (IFN-γ, IL-10, and TGF-β) of the supernatants was measured using Bioscience ELISA kits (BD Biosciences, San Diego, CA) after the manufacturer's instructions. Markers of the different lymphocyte and activation populations were detected using FITC-labeled anti-CD4, CD8 and CD86 antibodies (eBioscience, San Diego, CA) and flow cytometry (Flow cytometer EPICS® XL-MCL; Beckman Coulter, Florida) . The implication of the nuclear factor (NF) kB mediated signal transduction pathway in the immune response was determined by the addition of an inhibitor (lactacystin).

Results

Feces from patients with celiac disease, who presented alterations in the composition of the microbiota, induced a higher production of pro-inflammatory cytokines (TNF-α and IFN-γ) than healthy individuals and a production of the lower anti-inflammatory cytokine IL-10 to healthy individuals in PBMC. These also increased the synthesis of the essential CD86 surface molecule for T cell activation more than the control feces. Co-incubation with bifidobacteria regulates the proinflammatory cytokine profile induced by the fecal microbiota of patients with celiac disease, reducing the synthesis of TNF-α and IFN-γ and increasing that of IL-10. Inhibition of cytokine synthesis in the presence of lactacystin suggests that NF kB is involved in the immunomodulatory effects exerted by bifidobacteria. 53 ΕΡ 2236598Β1

EXAMPLE 8 CHARACTERIZATION OF FRACTIONS OF GLIADINES PRODUCED IN THE IN VITRO DIGESTION PROCESS AND CHARACTERIZATION OF THE REDUCTION OF PEPTIDES DERIVED FROM THE GASTROINTESTINAL DIGESTION OF GLIADINES BY BIFIDOBACTERIA AND THEIR BIOLOGICAL EFFECTS 1. Digestion of Gliadin Gastrointestinal

A preparation of gliadins (Sigma, G3375) containing four of the isoforms, α-β-, γ-, and ω- gliadins of these proteins whose complete amino acid sequence is available is used. The solution of different aliquots (150 mg) of commercial gliadin extract was performed in isotonic saline solution (140 mM NaCl, 5 mM KCl) at pH 3, heating the mixture at 55øC for 30 minutes in a stirred bath. Samples were subjected to a simulated gastrointestinal digestion procedure using pepsin (Sigma, P7000) (800-2500 IU / mg protein) and porcine pancreatin (P1750) (activity, 43USP), and bile extract (B3883). Gastric digestion (pepsin in 0.1 M HCl / pH 3/1 h, with shaking) was performed in centrifuge tubes (50 ml). Subsequently, the intestinal stage (pancreatin-bile in 0.1 M NaHC03 / pH 6.9-7 / 2h, with shaking) was performed in the upper (donor) compartment (1.5 ml) of a double chamber system prepared with a dialysis membrane (Spectra / Por 2.1, Spectrum Medicai, Gardena, CA) having a pore size of 15 KDa. A saline solution (1 mL) was placed in the lower compartment (receiver). Upon completion of the digestion process, the total protein content was quantified in the supernatant (4000 rpm / 5 min / 4 ° C) of the digested gastrointestinal sample and the dialysed sample using a commercial kit (Sigma, TP0200) based on the method of Lowry. 2. Chromatographic analysis of gliadin fractions after the in vitro digestion process.

To analyze the gliadins, a reverse phase chromatographic method was adapted. The separation was performed on a BioBasic C18 column (5 mm 4.6x250 mm) using the Hewlett Packard 1050 HPLC equipment. The mobile phases used consist of (A) 15% (v / v) aqueous Acetonitrile (ACN, HPLC grade) with 0.1% (v / v) Trifluoroacetic acid (TFA), and (B) 80% (v / v) with 0.1% TFA (v / v). The elution gradient used was as follows; 0-5 min, linear gradient up to 5% of solvent B; 5-12 min, linear gradient to 20% solvent B. The column was washed with 100% solvent B for 5 minutes, and was rebalanced using the initial conditions for 3 minutes. Samples were filtered through a nylon membrane (13 mm 0.22 mm Millex GN, Millipore). Three independent aliquots of each treatment were analyzed (100 μΐ). Absorption in the ultraviolet region was monitored at 210 nm. 3. Identification of gliadin peptide sequences using reverse phase chromatography coupled to electrospray-MsMs (RP-HPLC-ESI-MS / MS).

The chromatographic separation was performed on a BioBasic C18 column (5 mm 4.6 x 250 mm) using an HPLC system

Agilent coupled to a quadrupole ion trap mass spectrometer (Esquire-LC-Ms (n), Bruker Daltonics, Billerica, MA). The chromatographic elution gradient employed was that described in the previous section. In the analysis the nitrogen was used as nebulizer and drying gas, and helium as the gas for the molecular collision at a pressure of approximately 5 x 10 3 bar. The capillary voltage of the collision was maintained at 4 kV. The mass spectrum was

monitored in the 500-5000 mass / charge range. The method shows the mean mass (Ms) analysis of the spectrum 15, and the Ms (n) sequential mass analysis of the spectrum 5. The ion current limit for effecting the sequential mass analysis was set to 5000, and the precursor ions were isolated in a m / z range of 4.0 fragmented with a voltage ramp of 0.39 to 2.6 V. The m / z spectral data were processed and transformed using the computer analysis software provided by the manufacturer (Data Analysis version 3, Bruker Daltonics). The peptide sequences of the Ms (n) spectrum were established using computational analysis software (BioTools version 2.1 (Bruker Daltonics).) 4. Effects of co-incubation of gastrointestinal gliadins digested with bifidobacteria.

The digested gastrointestinal gliadin samples obtained in section 1 were co-incubated in the presence and absence of intestinal bifidobacteria, such as the strain of the invention, located in the apical part (donor) of a transposable Caco-2 cell culture, used as template of the intestinal epithelium. In order to estimate the reduction of the toxic effects of gliadins in the presence of the strain of the invention (IATA-ES1), the synthesis of the proinflammatory cytokines TNF-α and NFκB was measured using ELISA methods in the Caco-2 cell culture of the chamber lower 56 ΕΡ2236598Β1 (receptor) in the presence and absence of bifidobacteria (Figures 3 and 4).

Results

RP-HPLC analysis of the protein fraction below 15 KDa derived from the digestion of gastrointestinal gliadin samples digested. Chromatographic separation (Figure 2) reveals the presence of five peptide fractions being the major fraction # 2. Dialysis studies of gliadin-derived peptides in the presence of different bacterial strains (ie, strain of the invention (IATA-ES1) and Bifidobacterium A2, isolated in our laboratory demonstrated the proteolytic capacity of these bacteria in gliadins and specifically in fraction # 2. strain that caused the most significant reduction of fraction # 2 was the strain of the invention (IATA-ES1), which reduced more than 10% of the fraction under the study conditions. EXAMPLE 9. ASSESSMENT OF CAPACITY OF BIFIDOBACTERIUM GENDER STRENGTHS REGULATE MATURATION INDUCED BY GLIADINE AND PRO-INFLAMMATORY FEOTYPE IN DENDRITIC CELLS INVOLVED IN THE PRESENTATION OF ANTIGEN 1. Preparation of cultures of intestinal bifidobacteria and fecal samples for evaluation.

Strains of the genus Bifidobacterium were inoculated into 10 ml of MRS growth medium (Scharlau Chemie SA, Barcelona, Spain) containing 0.05% cysteine (MRS-C) at 1% with a 24 h culture and incubated for 22 h 37 ° C in anaerobiosis (AnaeroGen, Oxoid, Basingstoke, UK). The 57 Ρ 2236598 Β 1 cells were collected by centrifugation (6000 g, 15 min), washed twice in PBS (10 mM sodium phosphate, 130 mM sodium chloride, pH 7.4), and resuspended in PBS containing 20. % glycerol. Aliquots of these suspensions were frozen using liquid nitrogen and stored at -80 ° C. The number of viable cells after the freeze-thaw cycle was determined by counting on MRSC agar plates after incubation for 48 h. Viability was greater than 90% and all cases. Each aliquot was used for a single test. Before using the cells as a stimulus these were washed by centrifugation and resuspended in PBS. 2. Isolation and stimulation of dendritic cells (DC) obtained from peripheral blood.

PBMC were isolated from the peripheral blood of 4 healthy volunteers (mean age 30 years, ranging from 24-40) in heparin tubes. PBMCs were isolated by Ficoll gradient centrifugation (Amersham Biosciences, Piscataway, NJ). The cells were washed with RPMI 1640 medium (Cambrex, New York, USA) and adjusted to a density of 1 x 106 cells / ml in RPMI 1640 medium also containing 10% fetal bovine serum (Gibco, Barcelona, Spain), 2 mM L-glutamine, 100 pg / ml streptomycin and 100 U / ml penicillin (Sigma). PBMCs were incubated in 24-well flat-bottomed polystyrene plates (Corning, Madrid, Spain) in the presence or absence of stimulation agents at 37øC, 5% ot for 24 h. as a stimulus was used gliadin (0.1 pg / ml) and IFN-γ (150 IU) in the presence and absence of bifidobacteria and other intestinal bacteria. As a positive control, purified E. coli 0111: B4 (Sigma, St. Louis, MO) purified Î »22365981 lipopolysaccharide (LPS) at a concentration of 1 mg / ml was used. As a negative control cytokine production in unstimulated PBMC was tested. Each stimulus type was tested in duplicate in each experiment. Culture supernatants were collected by centrifugation, fractionated and stored in aliquots at -20 ° C until detection of the cytokine. 3. Determination of cytokine markers and cellular activation

Cytokine (IFN-γ, IL-10, and TGF-β) concentrations of the supernatants were measured using Bioscience ELISA kits (BD Biosciences, San Diego, CA) following the manufacturer's instructions. Activation molecule markers were detected using FITC-labeled HLA-DR, CD86, CD40 and CD83 antibodies (eBioscience, San Diego, CA) and quantified by flow cytometry (Flow cytometer EPICS® XL-MCL; Beckman Coulter, Florida).

Results The strain of the invention (IATA-ES1) reduced at least 10% the production of IFN-γ, the major inflammatory cytokine that is produced in response to gliadins in patients with celiac disease, caused by stimulation of dendritic cells with gliadins and other potentially proinflammatory intestinal bacteria (eg bacteroids and enterobacteria isolated from patients with celiac disease). This strain also produced an increase in the synthesis of the anti-inflammatory cytokine IL-10 by dendritic cells of at least 200% compared to the effect produced by the stimulation with 59 Ρ 2236598Β1 gliadins. The strain of the invention (IATA-ES1) reduced the expression of HLA-DR and CD86 molecules, induced after incubation of the dendritic cells in the presence of gliadin and IFN-γ, between 15 and 20%.

Lisbon, March 25, 2013 60

Claims (15)

1. Strain of Bifidobacterium longum deposited in the Colección Española de Culturas Tipo (CECT) with access number CECT 7347. A strain as claimed in claim 1, in the form of viable cells. A strain as claimed in claim 1, in the form of non-viable cells. A combination of microorganisms, comprising the strain as claimed in any of claims 1 to 3 and at least one other microorganism. A combination of microorganisms as claimed in claim 4, wherein the other microorganisms are B. longum ATCC 15707 and / or L. lactis NCD0712. A supernatant of a culture or extract obtained from the microorganism as claimed in any of claims 1 to 3, or the combination of microorganisms as claimed in claims 4 or 5. Use of the B. longum strain as claimed in any one of claims 1 to 3; or the combination of microorganisms as claimed in claims 4 or 5; or supernatants or extracts as claimed in claim 6, for the preparation of formulations designed to reduce risks and improve the health status of patients with gluten-related diseases. 1 ΕΡ 2236598Β1 Use as claimed in claim 7 for the preparation of formulations designed for the prevention and / or treatment of celiac disease. Use as claimed in claims 7 or 8, wherein the formulated formulation is a food, a nutraceutical, a supplement, a pharmaceutical composition, a probiotic and / or a symbiotic. A nutritional composition comprising the B. longum strain as claimed in any one of claims 1 to 3, or the combination of microorganisms as claimed in claims 4 or 5, or the supernatants or extracts as claimed in claim 6. A composition as claimed in claim 10, which further comprises a carrier. A composition as claimed in claim 11, wherein the carrier is milk, yoghurt, cheese, fermented milk, dairy products, cereals, fermented cereals, juices, ice creams, and / or formulations for children. A composition as claimed in any of claims 10 to 12, wherein the B. longum strain is present in an amount of about 106 cfu to about 109 cfu per gram or milliliter of the composition. A composition as claimed in any one of claims 10 to 13, wherein said composition can be found in the form of a tablet, capsule, microcapsule, powder, solution or paste. 2 ΕΡ 2236598Β1 A pharmaceutical composition comprising the B. longum strain as claimed in any one of claims 1 to 3 or the combination of microorganisms as claimed in claims 4 or 5; or the supernatants or extracts as claimed in claim 6, together with appropriate amounts of pharmacologically acceptable excipients. Lisbon, March 25, 2013